Antigen-drug vehicle enabling transmucosal and transdermal administration, and method of inducing mucosal immunity and mucosal vaccine and DDS using the same

ABSTRACT

Disclosed are an antigen-drug vehicle (AD vehicle) exerting a function of inducing the preferential and selective production of an antigen-specific secretory IgA antibody, local immunity or mucosal immunity; a method of inducing mucosal immunity, a mucosal vaccine and a preventive or a remedy for allergy using the above AD vehicle; and a transmucosal or transdermal DDS using the above vehicle.

This application is a U.S. national stage of International ApplicationNo. PCT/JP2005/005659 filed Mar. 22, 2005.

TECHNICAL FIELD

The present invention relates to an antigen-drug vehicle enablingtransmucosal and transdermal administration, and more specifically, to amethod of inducing mucosal immunity which causes preferential andeffective, particularly selective production of an antigen-specificsecretory immunoglobulin A, a mucosal vaccine, prevention or treatmentof allergy, and a drug delivery system, which is characterized by usingthe vehicle for desired antigen or drug.

BACKGROUND ART

Conventional inactivated vaccine, toxoid, and the like are known to havethe following drawbacks:

(1) Lack of Defense Against Infection in Natural Infection Route

The natural infection route of bacterial, virus, and the like is, forexample, the mucosal membrane of the nasal cavity, the trachea, theintestinal tract, and the like, whereas the vaccine inoculation route issubcutaneous, intramuscular, and the like, which is different from theroute described above. It is desired to achieve defense againstinfection by an inoculation route fit for the actual conditions of thenatural infection, particularly defense against infection at the mucosalmembrane from vaccine administration via the mucosal membrane.

(2) Low Mucosal Immunity

In the subject for vaccine inoculation, immunoglobulin G (hereinafter,simply referred to as “IgG” or “IgG antibody”) is mainly produced intothe blood, and humoral immunity is induced. However, secretoryimmunoglobulin A (hereinafter, simply referred to as “IgA” or “IgAantibody”) which takes charge of mucosal immunity, is nearly notproduced, and therefore, establishment of mucosal immunity is notexpected. Furthermore, necessity and effectiveness of IgA antibody areas follows: IgA antibody plays a very important role in clinicalimmunity by taking charge of mucosal immunity, i.e., defense againstinfection at the mucosal membrane which is the entrance to infectiononto the respiratory organs such as the nasal cavity and the trachea byspray or air, and infection onto the intestinal tract by oral.Furthermore, IgA antibody has cross immunity, i.e., cross-neutralizationactivity, and broad spectrum for infection defense as much, and also hasdefense against infection for variant antigen, whereas IgG antibody hashigh specificity for antigen, but has narrow spectrum for infectiondefense, leading to nearly not being effective for infection defenseagainst variant antigen.

(3) Necessity of Additional Vaccination and Added Expense

Since IgG antibody is produced low only by one injection of the firstimmunization, and certain effects cannot be expected, it is required toincrease blood IgG antibody value by additional inoculation, so-calledbooster inoculation of once or more times, based on the conditions ofIgG antibody retention later. Therefore, repeated expense and effort arerequired, and on top of that, the case is often found where the chanceof the booster inoculation is not effective for young children,especially infants two-years old or less, who is likely to depart fromthe chance of the booster inoculation, though the effects are recognizedfor the elderly, adults, and school children, who are benefited from thechance.

In other words, conventional inactivated vaccine, toxoid, and the likeinduces mainly production of blood IgG antibody in the subject forvaccine inoculation, and also brings actions and effects of increasinghumoral immunity, being recognized for its efficacy. However, it has lowperformance of inducing IgA antibody production or mucosal immunity,showing the limit about sufficient function and effects for defenseagainst natural infection. From such circumstances, there have been manytrials from various sides so far to resolve the drawbacks of theconventional vaccines. For example, it includes improvement of thevaccine antigen in quality or quantity, experimental production of livevaccine replacing inactivated vaccine, development of new inoculationroute, mucosal vaccine, and the like, screening of adjuvant, whichelevates humoral immunity and cause maintenance thereof, development ofmucosal immunity adjuvant, and the like. However, development of mucosalvaccine which is safe and effective has not been achieved.

Hereinafter, development of mucosal vaccine will be explained.

(1) Increase of the Amount of the Vaccine Antigen

A trial has been conducted of increasing the amount of the vaccineantigen which is inoculated subcutaneously or intramuscularly, orincreasing the amount of IgG and IgA antibodies, which are secreted tothe mucosal membrane. For example, a method wherein neuraminidase of thevirus membrane protein is added to and mixed with conventionalinactivated influenza vaccine to increase antibody production amount, ora method wherein MF59 is added and mixed as an adjuvant, and the likehas been tried. However, these methods have been found to havedisadvantages such as incurrence of pain, strong adverse reaction, andthe like.

(2) Vaccine of Nasal Administration Type

A method has been tried wherein liquid split antigen is directlyinoculated nasally for infection defense by IgA antibody, which isconsidered most effective, but the fact is pointed out that IgAproduction amount is small. In order to elevate IgA antibody productionability, there has been a trial that Escherichia coli heat-labile livetoxin or cholera toxin is added to and mixed as an adjuvant with thesplit antigen to elevate mucosal immunity response, i.e., IgA antibodyproduction ability. However, from the circumstances that the safety ofthe toxin as an adjuvant has been not proved, the trial treatment hasbeen stopped, and not been put to practical use.

(3) Live Vaccine Using Cold-Attenuated Strain Which Can be InoculatedInto the Nasal Cavity

A method is put to practical use wherein cold-attenuated influenza virusstrain, which has optimal growth temperature of 25° C., and does notgrow at 39° C., but the risk of toxicity recovery cannot be denied asthe mechanism of attenuation of the parent cold-attenuated strain is notclear. In addition, since the active ingredient of the vaccine is a livevirus, it has high invasion force into cells and excellent ininitialization of immunity, but mild influenza symptoms often incur, sodefects have been found that it cannot be used for human who has highrisk of increase in severity when infected by influenza, elderly people,and the like.

(4) Other Vaccines

Developments of a vector vaccine which has vaccinia virus as a virusvector, attenuated live vaccine by reverse genetics, DNA vaccine whichuses DNA or cDNA as an active ingredient, have been in progress in lablevel, but not put to practical use.

Furthermore, development of immunity adjuvant will be explained below.

(1) Immunity Adjuvant

The immunity adjuvant is a general name of a substance which hasregulation activity such as reinforcement or inhibition of immuneresponse, and largely divided into two kinds: a substance which isrelated to a dosage form for the purpose of sustained-release,retention, and the like of antigen within the subject for inoculation,and a substance which helps elevation, inhibition, and the like ofimmune response. Between them, as the former, i.e., as the adjuvant fordosage form, for example, vaccine or toxoid with use of aluminumphosphate, alum, and the like has been already put to practical use.However, the latter, i.e., the adjuvant which helpsreinforcement/elevation of immune response has not been known yet to beput to practical use. For example, BCG live bacteria derived frombacteria, BCG-CWS, endotoxin, glucan, and the like, synthesized MDP,levamisole, Poly I-Poly C, bestatin, and the like, and interferons suchas cytokines, TNF, CSF, and the like have been known, but it isconsidered that guarantee for the safety and efficacy is needed for thepractical use of them, by the reasons of insufficient effects, and thelike for adjuvant diseases such as arthritis, chronic arthriticrheumatism, hyper-γ-globulinemia, anemia, and the like. In addition, atechnique is known (Patent Document 1) using a pulmonarysurfactant/protein derived from a higher animal as an adjuvant in orderto enhance induction of humoral immunity, but it has not known to be putto practical use.

(2) Development of Adjuvant for Mucosal Immunity

Various adjuvants have been developed, for example, pertussis toxin Boligomer (Patent Document 2), cholera toxin (Patent Document 3),Escherichia coli heat-labile enterotoxin B subunit (LTB) (PatentDocument 4), starch particles (Patent Document 5), cholera toxin B chainprotein (CTB) (Patent Document 6), B subunit of verotoxin 1 (PatentDocument 7), oligonucleotide (Patent Document 8), interleukin 12(Non-Patent Document 1), and the like. However, they have not been putyet to practical use.

As described above, necessity has been recognized widely and deeply forreplacement of the conventional vaccine which is inoculatedsubcutaneously, intramuscularly, and the like with a mucosal vaccinewhich induces production of IgA antibody in the mucosal membrane, whichis a natural infection route of virus. Particularly, as a vaccine ofnext generation in the 21 century, so-called mucosal vaccine, whichinduces production of IgA antibody, and local immunity or mucosalimmunity, is expected and hoped worldwide to be developed and put topractical use, but not yet achieved. The reason is considered to be inthe fact that safe and effective adjuvant, which imparts the function ofinducing production of IgA antibody, and local immunity or mucosalimmunity to vaccine, has not specified or established.

-   Patent Document 1: JP-T-2002-521460-   Patent Document 2: JP-A-3-135923-   Patent Document 3: JP-T-10-500102-   Patent Document 4: JP-T-2001-523729-   Patent Document 5: JP-T-2002-50452-   Patent Document 6: JP-A-2003-116385-   Patent Document 7: JP-A-2003-50452-   Patent Document 8: The pamphlet of PCT WO 00/20039-   Non-Patent Document 1: pp. 4780-4788, vol. 71, 2003, Infection and    Immunity-   Non-Patent Document 2: pp. 2-11, vol. 10, 2004, Journal of neonatal    Nursing-   Non-Patent Document 3: pp. 9-14, vol. 74 (suppl. 1), 1998, Biology    of the Neonate-   Non-Patent Document 4: pp. 452-458, vol. 24, 2001, American Journal    of Respiratory Cell and Molecular Biology

DISCLOSURE OF THE INVENTION

The present invention has the following objects: specifically, to impartthe function of inducing production of IgA antibody, and local immunityor mucosal immunity; development of safe and effective techniquetherefor; conversion from conventional humoral immunity vaccine to safeand effective mucosal immunity vaccine; prevention and treatment ofallergy; and establishment of transmucosal/transdermal drug deliverysystem (hereinafter, simply referred to as “DDS”) for administration andtransport of a drug via the mucosal membrane or skin.

As means to solve the objects, the present invention provides anantigen-drug vehicle enabling transmucosal and transdermaladministration, and a method of inducing mucosal immunity which causespreferential and effective, particularly selective production of anantigen-specific secretory Immunoglobulin A, a mucosal vaccine, an agentfor prevention or treatment of allergy, and a transmucosal ortransdermal DDS, which is characterized by using the vehicle for desiredantigen or drug.

Application or general use of the antigen-drug vehicle provided by thepresent invention, brings actualization and popularization of a mucosalvaccine against various infections, an agent for prevention or treatmentof allergy, and a transmucosal or transdermal DDS. The mucosal vaccine,which is an immunity means fit for the actual conditions of the naturalinfection, exerts remarkably excellent infection defense effects ascompared to the conventional vaccines. In addition, the nasalcavity-mucosal IgA induced by the antigen-drug vehicle, bringsinactivation of allergen, which makes reduced sensitization possible.Furthermore, application of aforesaid DDS for various drugs reinforcesand promotes preventive and/or therapeutic effects of drugs bytransmucosal and transdermal administration. As a result, this inventionhighly improves medical treatment, health and hygiene of the wholemankind, and also is hopeful good news to those engaged in the field ofmedical treatment, health and hygiene of the world. In addition, itprovides widely means of imparting function and performance which makesit possible transmucosal and transdermal administration, which is simplecompared to injection, widely for a biological preparation containingconventional and future vaccine, toxoid, and the like, and variousdrugs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents inhibition effects of influenza virus infection in (c)the nasal cavity and (d) the alveoli in (a) the cavity and (b) thealveoli of various nasal influenza vaccine administrations, andsubcutaneous-injection influenza vaccine administration. * representsthe significance level from the group of vaccine-independent (without ADvehicle or adjuvant) administration by T-test (p<0.01). (Example 1)

FIG. 2 represents the amounts of IgA and IgG, which producesanti-influenza antibody in the nasal cavity-washing solution byinfluenza vaccine of (a) nasal administration and (b) subcutaneousinjection. The white bar represents IgA amount, and the black barrepresents IgG amount, respectively. (Example 2)

FIG. 3 represents the influence of the adjuvant on production ofanti-influenza specific antibodies, IgA and IgG in the lung-washingsolution by influenza vaccine of (a) nasal administration and (b)subcutaneous injection. (Example 3)

FIG. 4 represents the influence of PSF-2 and CTB on production of bloodanti-influenza specific antibodies, IgA and IgG by influenza vaccine of(a) nasal administration and (b) subcutaneous injection. (Example 4)

FIG. 5 represents the influence of PSF-2 and CTB on the secretion levelof TGF-β1 in the mucosal membranes of (a) the nasal cavity and (b) thealveoli by nasal administration of influenza vaccine. (Example 6)

FIG. 6 represents the influence of PSF-2 and CTB on production ofanti-influenza specific antibody in (a) the nasal cavity, (b) thealveoli and (c) the blood by nasal administration of influenza vaccine.(Example 7)

FIG. 7 represents the influence of SPF-2 and CTB on production ofvarious cytokines which is secreted from the nasal cavity, the alveoliand lymphocyte of the spleen by nasal administration of influenzavaccine. (Example 8)

FIG. 8 represents the influence of PSF-3 on production of anti-influenzaspecific antibody in (a) the nasal cavity, (b) the alveoli and (c) theblood by nasal administration of influenza vaccine. (Example 9)

BEST MODE FOR CARRYING OUT THE INVENTION

Embodiments of the present invention will be explained below.

1. Explanation for Terms and Ingredients of the Antigen-Drug Vehicle

(1) Antigen-Drug Vehicle

The vehicle (Antigen-drug vehicle, hereinafter, simply referred to asthe “AD vehicle” or “ADV”) is a complex of lipid and protein which isdesigned to enable transmucosal administration and transdermaladministration of antigen, drug, and the like. The AD vehicle comprises(a) to (c) below.

(a) Pulmonary surfactant protein B or its fragment (including not onlynatural fragment obtained by protease, but also artificial fragmentobtained by genetic engineering or peptide synthesis, or variantfragment by substitution and/or deletion of at least one amino acidconstituting such fragment).

(b) Pulmonary surfactant protein C or its fragment (including not onlynatural fragment obtained by protease, but also artificial fragmentobtained by genetic engineering or peptide synthesis, or variantfragment by substitution and/or deletion of at least one amino acidconstituting such fragment).

(c) Lipid such as phospholipid and fatty acid. The shape and thestructure is a membrane form (lipid membrane of sheet form or rollingform) retaining polypeptide chain of thorn shape or spike shape, whichis a shape wherein the ends of the hydrophobic area of the multiplepolypeptide chains are intrusive into the lipid membrane to be implantedin spike shape, and different from conventional lipid vesicle(liposome).

If desired antigen, drug, and the like is subjected to coexistence,contact, capture, adhesion or binding (riding) with the antigen-drugvehicle according to the present invention, it makes it possibletransmucosal administration and transdermal administration of suchantigen, drug, and the like. In other words, aforesaid vehicle is a rideof them, enabling transmucosal administration and transdermaladministration of such antigen, drug, and the like.

In addition, ingredients of the AD vehicle, proteins, polypeptide,peptide or lipid used in preparation/production of the vehicle, i.e.,pulmonary surfactant proteins B and C, a fragment thereof, a variantfragment by substitution and/or deletion of at least one amino acidconstituting such fragment peptide, and the like, and lipid such asphospholipid, fatty acid, and the like will be explained in detaillater.

(2) Pulmonary Surfactant

The pulmonary surfactant has been put to practical use from the middle1990s in treatment of respiratory distress syndrome (RDS), andcurrently, various preparations derived from human, cattle, pig, and thelike have been marketed widely to be in practical use (Non-PatentDocument 2). In addition, synthetic peptide preparation containingactive domain related to RDS treatment has been also marketed, anddesign development or synthesis of SP-B and SP-C analogues have been inprogress (Non-Patent Document 3). Composition and constitution of thepulmonary surfactant are as follows: a complex comprising about 90% oflipid (67.3% of phosphatidyl choline, 19.3% of phosphatidylglycerol,3.2% of phosphatidyl serine, and other free fatty acids, and the like),and about 10% of protein (surfactant proteins A, B, C and D:Hereinafter, simply referred to as “SP-A”, “SP-B”, “SP-C” and “SP-D”,respectively). The molecular weight is 28 to 36 kDa for SP-A, 15 kDa forSP-B, 3.5 kDa for SP-C and 43 kDa for SP-D. SP-A and SP-D arehydrophilic (water-soluble) and lectin-like (membrane-associated). SP-Band SP-C are hydrophobic (lipid-soluble) and lipid-bindable, and hasintrusion ability into phospholipid membrane and surfactant action. Thepulmonary surfactant protein genes derived from human, cattle, pig, andthe like are known, and for example, the accession number of full-lengthbase sequence of human SP-B gene DNA is J02761, and that of human SP-C(and SP-C1) is J03890 in GenBank/NCBI (http//www.ncbi.nlm.nih.gov./).The coding region (CDR) of the human SP-B and SP-C obtained from NCBIand the amino acid sequence encoded by them, are described below.

SEQ ID No. 1: CDR base sequence of human SP-B gene DNA;

SEQ ID No. 2: Human SP-B full-length amino acid sequence interpretedfrom SEQ ID No. 1;

SEQ ID No. 3: CDR base sequence of human SP-C gene DNA;

SEQ ID No. 4: Human SP-C full-length amino acid sequence interpretedfrom SEQ ID No. 3;

SEQ ID No. 5: CDR base sequence of SP-C1 located on human SP-C gene DNA;and

SEQ ID No. 6: Human SP-C1 full-length amino acid sequence interpretedfrom SEQ ID No. 5.

(3) Protein or Peptide Used in This Invention

In preparation/production of the antigen-drug vehicle related to thepresent invention, combination of SP-B and SP-C, and combination of SP-Band SP-C1, which are derived from mammals such as human, cattle, pig,whale, dolphin, and the like, or fishes such as tunny, shark, ray,yellowtail, and the like, can be used respectively. For example, use canbe also made of combination of SP-B and SP-C and combination of SP-B andSP-C1, which are human-derived proteins comprising the full-lengthamino-acid sequence described in SEQ ID Nos. 2, 4 and 6, respectively,can be used respectively. Furthermore, for example, use can be also madeof hydrophobic (lipid-soluble) region of SP-B and SP-C based on thehydrophobicity value of Kyte-Doolittle and a fragment comprising theregion, a variant fragment by substitution and/or deletion of at leastone amino acid constituting such fragment peptide. For example, use canbe made of natural peptide or peptide obtained by genetic engineering orchemical synthesis comprising the amino acid sequence described in SEQID Nos. 7 to 20, long chain peptide from them comprising such peptide,and a variant or synthetic analogue by substitution and/or deletion ofat least one amino acid constituting such peptide. The amino acid numberis represented by the ordinal number in the order from Met occupying theN-end of each sequence as first amino acid, toward the C-end direction(direction from the left to the right of the described sequences).

SEQ ID No. 7: Amino acid sequence of No. 214 to No. 225 of SEQ ID No. 2(SP-B fragment);

SEQ ID No. 8: Amino acid sequence of No. 257 to No. 266 of SEQ ID No. 2(SP-B fragment);

SEQ ID No. 9: Amino acid sequence of No. 29 to No. 58 of SEQ ID Nos. 4and 6 (SP-C fragment);

SEQ ID No. 10: Amino acid sequence of No. 1 to No. 20 of SEQ ID No. 2(SP-B fragment);

SEQ ID No. 11: Amino acid sequence of No. 102 to No. 110 of SEQ ID No. 2(SP-B fragment);

SEQ ID No. 12: Amino acid sequence of No. 119 to No. 127 of SEQ ID No. 2(SP-B fragment);

SEQ ID No. 13: Amino acid sequence of No. 136 to No. 142 of SEQ ID No. 2(SP-B fragment);

SEQ ID No. 14: Amino acid sequence of No. 171 to No. 185 of SEQ ID No. 2(SP-B fragment);

SEQ ID No. 15: Amino acid sequence of No. 201 to No. 279 of SEQ ID No. 2(SP-B fragment);

SEQ ID No. 16: Amino acid sequence of No. 253 to No. 278 of SEQ ID No. 2(SP-B fragment);

SEQ ID No. 17: Amino acid sequence of No. 300 to No. 307 of SEQ ID No. 2(SP-B fragment);

SEQ ID No. 18: Amino acid sequence of No. 317 to No. 330 of SEQ ID No. 2(SP-B fragment);

SEQ ID No. 19: Amino acid sequence of No. 344 to No. 351 of SEQ ID No. 2(SP-B fragment);

SEQ ID No. 20: Amino acid sequence of No. 358 to No. 381 of SEQ ID No. 2(SP-B fragment);

SEQ ID No. 21: Amino acid sequence of No. 24 to No. 58 of SEQ ID Nos. 4and 6 (SP-C fragment).

According to the present invention, use can be made of combination of atleast one kind selected from a group consisting of SP-Bs comprising theamino acid sequences described in SEQ ID Nos. 2, 7, 8 and 10 to 20, anda fragment thereof, and at least one kind selected from a groupconsisting of SP-Cs (and SP-C1) comprising the amino acid sequencesdescribed in SEQ ID Nos. 4, 6, 9 and 21, and a fragment thereof.

(4) Lipid Used in the Present Invention

As the phospholipid, phospholipid containing the pulmonary surfactant,for example, phosphatidyl choline (lecithin), dipalmitoyl phosphatidylcholine, phosphatidyl serine, and the like is preferably used.Additionally, use can be made of dipalmitoyl glycerophosphocholine,diacyl glycerophosphoglycerol, phosphatidylglycerol (cardiolipine),dilauroyl phosphatidylglycerol, dimiristoyl phosphatidylglycerol,dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol,phosphatidyl inositol, phosphatidyl ethanolamine, phosphatidic acid,sphingomyelin, and the like. In addition, as the fatty acid, use can bemade of lauric acid, myristic acid, palmitic acid, stearic acid,palmitoleic acid, oleic acid, and the like. In addition, use can be madeof lipid derived from aquatic animal such as whale, tunny, dolphin, andthe like, which has active pulmonary expansion.

(5) The Pulmonary Surfactant Preparations in the Market for RDSTreatment

According to the present invention, the pulmonary surfactantpreparations in the market, for example, Surfacten (trademark),Infasurf, Curosurf, Humansurf, Exosurf, Alveofact, and the like can beused as the AD vehicle, which have been approved by the relevantauthority for the safety and efficacy as a RDS treatment drug, andcontains hydrophobic or lipid-soluble SP-B and SP-C and phospholipid. Inaddition to SP-B and SP-C, the marketed preparations containinghydrophilic or water-soluble SP-A and SP-D can be given to use afterextracting the water-soluble proteins SP-A and SP-D with 1-butanol, andthe like, and removing them to the detection limit or less. In addition,use of dry preparation is more desirable than liquid preparationconsidering the adjustment of the concentration to be used inpreparation of the AD vehicle.

2. Novel Findings as a Base for the Present Invention

Under such very strict background techniques, this invention is made bykeen observation and analysis of the present inventors from trial anderror repeated over about 10 years, and profound knowledge andexperience and novel idea, and it is based on the following astonishingfindings.

(1) Conventional adjuvant reinforces antigen presenting-performance bycausing inflammation, and originally there are four kinds of proteinactive ingredients, SP-A, SP-B, SP-C and SP-D, which are surfactantssecreted by pulmonary or intestinal tract mucosal membrane. It has beenfound that if virus antigen is subjected to coexistence, contact,capture or adsorption with a complex of the combination of SP-B and SP-Cexcluding SP-A and SP-D, and phospholipid, or a complex of thecombination of synthetic peptides of both fragments of SP-B and SP-Ccomprising lipid-soluble region thereof (active region), and lipidmembrane (aforesaid AD vehicle), the antigen-presenting cells in themucosal membrane of the nasal cavity are activated without causinginflammation, virus antigens are incorporated into the cells in goodefficiency, and also anti-virus IgA production by the mucosal membraneis induced effectively and preferentially, and further selectivelywithout causing induction of IgG production in the mucosal membrane orblood.(2) In addition, it has been found that by adding to and mixing acomplex of the combination of SP-B and SP-C, and phospholipid, or acomplex of the combination of synthetic peptides of both fragments ofSP-B and SP-C comprising lipid-soluble region thereof (active region),and lipid membrane (aforesaid AD vehicle), with influenza virus splitantigen which is used as safe inactivated vaccine antigenconventionally, selective induction of secretory IgA production isachieved with the activation of the antigen-presenting cellssufficiently reinforced and supplemented, which is poor in independentsplit antigen compared with live vaccine, while maintaining high safetyof the split antigen.3. Process of Completing the Above-Mentioned Findings(1) The present inventors have studied extensively to elucidate themechanism of the development of influenza, and a treatment andprevention method for influenza. During the process, it has beenelucidated that pulmonary surfactant adsorbs and inactivates tryptaseclara of hemagglutinin (HA) processing protease of the respiratorytract, which manifests virus membrane infusion activity and infectiousability by restrictive cleavage of HA of the influenza virus membraneprotein, resulting in inhibiting the growth cycle of the virus.(2) As a result of continuous study, it has been found that in additionto the actions described above, the pulmonary surfactant selectivelyactivates mucosal antigen-presenting cells, and thus activates immunityperformance against virus antigen, leading to induction of secretoryIgA, but not leading to induction of IgG. Furthermore, it has been shownthat SP-B and SP-C are important as well as the lipid ingredient, asactive ingredients reinforcing mucosal immunity in the pulmonarysurfactant. The active region of these protein ingredients has beenspecified, and the efficacy of mucosal immunity reinforcement has beenevaluated.(3) Furthermore, studies have been progressed in view of a biologicaldefense substance in the mucosal membrane of the respiratory tract, anddefense against virus infection as described above, and it has beenproved that the pulmonary surfactant, which is secreted in the livingbody, is involved in selective induction of IgA production as mucosalimmunity adjuvant derived from the living body.(4) Studies have continued giving attention to the fact that (a) thepulmonary surfactant, which is originally physiologically activesubstance in the living body, has property of adsorbing specificbio-substance (Kido H., et al. FEBS Lett. Pulmonary surfactant is apotential endogenous inhibitor of proteolytic activation of Sendai virusand influenza A virus, 322 (29), 115-119, 1992), (b) the pulmonarysurfactant is secreted from alveolar type II cell or clara cell, andthen selectively incorporated into macrophage (Akira Suwabe, J. Jpn.Med. Soc. Biol. Interface; Surfactant metabolism disorder in alveolarproteinemia, 33, 10-13, 2002), and (c) the pulmonary surfactant isincorporated and metabolized in analogous cells, for example,antigen-presenting cells (dentritic cells).

As a result, it has been found that only SP-B and SP-C among the proteiningredients, and lipid ingredients of the pulmonary surfactant, functionas the “AD vehicle” of mucosal vaccine which selectively induces IgAproduction, and that the active ingredient region of SP-B or activedomain of mucosal immunity induction is a peptide comprising thefollowing amino acid sequences:

SP-B 214-225: (SEQ ID No. 7)Leu Ile Lys Arg Ile Gln Ala Met Ile Pro Lys Gly; and SP-B 257-266:(SEQ ID No. 8) Leu Leu Asp Thr Leu Leu Gly Arg Met Leu.

In addition, it has been found that the active ingredient region of SP-Cor active domain of mucosal immunity induction is a peptide comprisingthe following amino acid sequences:

SP-C 29-58: (SEQ ID No. 9)Cys Pro Val His Leu Lys Arg Leu Leu Ile Val ValVal Val Val Val Leu Ile Val Val Val Ile Val Gly Ala Leu Leu Met Gly Leu.(5) In addition, it has been found as the mechanism of selective IgAinduction that the active ingredients of the pulmonary surfactanteffectively conduct antigen presentation of T-lymphocyte by inducingincreased expression of MHC Class II, CD40 and B7-2 ofantigen-presenting dendritic cells, and in addition, promote classswitch to IgA-producing B-lymphocyte by inducing cytokine TGF-β1 of alocal mucosal membrane.4. The Objects of the Present Invention Completed Based on the Findingsand the Details Described Above are as Follows:(1) The first object is to establish a mucosal immunity method. Byproviding the “AD vehicle” and using it, the present invention achievesselective induction of production of antigen-specific IgA, which is anactive substance of mucosal immunity, and also establishes induction ofsafe and effective (with no adverse effects) mucosal immunity, and amethod thereof.(2) The second object is to improve qualities related to safety,efficacy and uniformity of the AD vehicle by use of a synthetic peptide.The present invention provides a complex (the AD vehicle), which isprepared with a synthetic peptide of active domain of SP-B mucosalimmunity induction (comprising each amino acid sequence of SP-B 214-225and SP-B 257-266 described above), a synthetic peptide of active domainof SP-C mucosal immunity induction (comprising each amino acid sequenceof SP-C 29-58 described above), a synthetic analogue thereof, along-chain synthetic peptide comprising these amino acid sequence as apart, and the like, and a lipid ingredient of the pulmonary surfactant,and improves the quality of the AD vehicle.(3) The third object is conversion of conventional subcutaneousinoculation of vaccine to mucosal administration. The present inventionemploys the AD vehicle in inactivated vaccine of respiratorytract-infection virus, for example, inactivated vaccine of influenza,SARS, the measles, rubella, mumps, and the like, and inactivated vaccineof intestinal tract-infection virus, for example, inactivated vaccine ofrotavirus, poliovirus, and the like, and converts subcutaneousinoculation vaccine thereof to mucosal vaccine.(4) The fourth object is to provide a method of using the AD vehicle ininactivated vaccine against virus infection via mucosal membrane exceptfor the respiratory tract and the intestinal tract, for example,inactivated vaccine against AIDS, Type B hepatitis, Type C hepatitis,and the like.(5) The fifth object is to provide a method of using the AD vehicle inDNA vaccine, live vaccine, prevention and treatment of allergy, and thelike.(6) The sixth object is to provide a method of using the AD vehicle intransdermal inoculation (application, pasting, and the like) as animmunity route which can induce IgA besides the mucosal membrane.(7) The seventh object is to open a way of use and application of the ADvehicle in agriculture, fishery, and the like, not only in DDS orpharmaceutics.

The AD vehicle provided by the present invention, has differentperformances and actions from adjuvants which have been usedconventionally in immunology as follows: Specifically, conventionaladjuvants are inoculated usually subcutaneously or intramuscularly,causing local inflammation reaction and attracting antigen-presentingcells or B- and T-lymphocytes, and has a foreign substance exerting itsability as active ingredient. In addition, they are used in combinationwith mineral oil or metal salt causing sustained-release and retentionof antigen to maintain inflammation reaction over long time. Inaddition, those known as conventional mucosal vaccine/adjuvants are asdescribed above, and a foreign substance such as Escherichia coliheat-labile live toxin and cholera toxin, and has risk of causingharmful actions or adverse actions. On the contrary, the AD vehicleaccording to the present invention, causes no local inflammationreaction. In addition, the AD vehicle is derived from biologicalingredient, and on top of that, it is limited to active ingredient inthe pulmonary surfactant or an active domain thereof, and useslow-molecular-weight peptide comprising such domain or domain region toachieve effective mucosal vaccine. Accordingly, the AD vehicle is verysafe and non-invasive.

5. According to the Present Invention, Following (1) to (5) areProvided, Respectively.

(1) An antigen-drug vehicle, which is a complex comprising at least onefragment selected from pulmonary surfactant protein B or multiplefragments derived from the protein B, at least one fragment selectedfrom pulmonary surfactant protein C or multiple fragments derived fromthe protein C, and at least one lipid.

More specifically, the AD vehicle is a complex comprising at least threesubstances in total, which are selected by at least one kind from eachgroup of following Group I (group of pulmonary surfactant protein B, andnatural and synthetic polypeptide derived or originated from the proteinB), Group II (group of pulmonary surfactant protein C, and natural andsynthetic polypeptide derived or originated from the protein C) andGroup III (group of lipid such as phospholipid, fatty acid, and thelike).

[Group I] Pulmonary surfactant protein B and a polypeptide comprisingfollowing amino acid sequence described in SEQ ID No. 2 (The amino acidnumber is assigned in the order from Met occupying the N-end of eachsequence as first amino acid, toward the C-end): Nos. 1 to 381 (SEQ IDNo. 2), Nos. 214 to 225 (SEQ ID No. 7), Nos. 257 to 266 (SEQ ID No. 8),Nos. 1 to 20 (SEQ ID No. 10), Nos. 102 to 110 (SEQ ID No. 11), Nos. 119to 127 (SEQ ID No. 12), Nos. 136 t 142 (SEQ ID No. 13), Nos. 171-185(SEQ ID No. 14), Nos. 201-279 (SEQ ID No. 15), Nos. 253-278 (SEQ ID No.16), Nos. 300-307 (SEQ ID No. 17), Nos. 317-330 (SEQ ID No. 18), Nos.344-351 (SEQ ID No. 19), NOs. 358-381 (SEQ ID No. 20), a polypeptidecomprising at least one sequence of the above-mentioned amino acidsequences as an active domain, a polypeptide by substitution and/ordeletion of at least one amino acid in the above-mentioned amino acidsequences, a synthetic analogue thereof, a modified body thereof bysaccharide or saccharide chain, and the like.[Group II] Pulmonary surfactant protein C and a polypeptide comprisingfollowing amino acid sequence described in SEQ ID No. 4 (The amino acidnumber is assigned in the order from Met occupying the N-end of eachsequence as first amino acid, toward the C-end): Nos. 1 to 197 (SEQ IDNo. 4), Nos. 29 to 58 (SEQ ID No. 9), Nos. 24 to 58 (SEQ ID No. 21), apolypeptide comprising the amino acid sequence of Nos. 1 to 191 of SEQID No. 6, a polypeptide comprising at least one sequence of theabove-mentioned amino acid sequences as an active domain, a polypeptideby substitution and/or deletion of at least one amino acid in theabove-mentioned amino acid sequences, a synthetic analogue thereof, amodified body thereof by saccharide or saccharide chain, and the like.[Group III] Lipid such as phospholipid like phosphatidyl choline,dipalmitoyl phosphatidyl choline, phosphatidyl serine, dipalmitoylglycerophosphocholine, diacyl glycerophosphoglycerol,phosphatidylglycerol, phosphatidyl inositol, phosphatidyl ethanolamine,phosphatidic acid, and the like, fatty acid like lauric acid, myristicacid, palmitic acid, stearic acid, oleic acid, and the like, and thelike.

In addition, the antigen-drug vehicle preferably has a shape andstructure wherein the above-mentioned Group III is a lipid membrane ofsheet form or rolling form, and multiple chains of each of theabove-mentioned Group I and Group II are implanted in spike shape withthe hydrophobic area ends being intrusive into the lipid membrane.

(2) A mucosal vaccine which is characterized by induction of mucosalimmunity, which is obtained by subjecting the antigen-drug vehicle ofthe above-mentioned (1) to coexistence, contact, capture or adsorptionwith antigen.

(3) An agent for prevention and treatment of allergy which ischaracterized by induction of mucosal immunity, which is obtained bysubjecting the antigen-drug vehicle of the above-mentioned (1) tocoexistence, contact, capture or adsorption with allergen. The actionand effects are by deactivation or reduced sensitization of allergensuch as cedar pollen coming by air and sucked, tick, and the like, bymucosal IgA in the nasal cavity or the nasal pharynx.(4) A transmucosal and/or transdermal DDS which is obtained bysubjecting the antigen-drug vehicle of the above-mentioned (1) tocoexistence, contact, capture or adsorption with a drug in an effectiveamount.(5) A method of inducing mucosal immunity which is characterized byadministering a mucosal vaccine, which is obtained by subjecting theantigen-drug vehicle of the above-mentioned (1) to coexistence, contact,capture or adsorption with antigen, into the nose or upper respiratorytract.

The induction of mucosal immunity in the inventions of theabove-mentioned (2), (3) and (5) is preferably characterized bypromoting the production of an IgA antibody in a local mucosal membrane,and further by promoting the production of TGF-β1 and Th2-type cytokinein the local mucosal membrane.

6. Hereinafter, Embodiments of the Present Invention Will be Explained.

(1) Composition of the AD Vehicle

Followings are dry wt. % of the three groups of the above-mentionedGroup I (group of pulmonary surfactant protein B, and natural andsynthetic polypeptide derived or originated from the protein B), GroupII (group of pulmonary surfactant protein C, and natural and syntheticpolypeptide derived or originated from the protein C) and Group III(group of lipid such as phospholipid, fatty acid, and the like): about0.1 to about 6.0 wt. % of Group I, about 0.1 to about 6.0 wt. % of GroupII and about 88 to about 99.8 wt. % of Group IIII. In preparation of theantigen-drug vehicle, the composition is adjusted to Group I %+Group II%+Group III %=100% by wt. %.

(2) Preparation of the AD Vehicle

Preparation procedures are as follows, for example: 2 mg of Group I, 2mg of Group II and 96 mg of Group III are weighed, respectively (Group I%+Group II %+Group III %=100% by wt. %), which are suspended uniformlyin 5 ml isotonic solution, for example, physiological saline solution orphosphate buffered saline (PBS). Resultant suspension is given to use asa solution of the antigen-drug vehicle (100 mg/5 ml). The vehicle isprepared in each use. For suspension, ultrasonic wave, homogenizer,mixer, shaker, and the like can be used. The ultrasonic wave is likelyto cause liquid change (increase of viscosity) by excessive treatment,and thus, a mixer, for example, box mixer (e.g., Vortex mixer(trademark)) is preferably used.

For specification of 96 mg of the lipid of Group III, for example, amixture of 71 mg of phosphatidyl choline, 21 mg of phosphatidylglyceroland 4 mg of phosphatidyl serine, and the like can be adopted (the totalof the lipid amount is 96 mg). In addition, in case of using marketedpulmonary surfactant preparation for RDS treatment, which is ensured tocontain SP-B and SP-C excluding SP-A and SP-D, the suspension preparedin accordance to the instruction manual, can be given to use as it is oras a solution of the antigen-drug vehicle.

(3) Preparation of Mucosal Vaccine

A mucosal vaccine is prepared by adding to and mixing the solution ofthe antigen-drug vehicle with an undiluted vaccine solution that thedry-weight ratio of the amount of the antigen-drug vehicle (V) to theamount of antigen in the vaccine (A) becomes about 0.2 to about 5. Forexample, if the antigen content is 1 μg/ml in 1,000 ml of the vaccineundiluted solution, and the weight ratio adopted is A/V=1, the additionand mixing amount of the solution of the antigen-drug vehicle (100 mg/5ml), which has been prepared in the above-mentioned (2), is 50 μl. Foruniform mixing, homogenizer, mixer, shaker, stirrer, and the like can beused.

(4) Preparation of Transmucosal or Transdermal DDS

The transmucosal or transdermal DDS can be prepared by using a druginstead of an antigen in the same manner as in the above-mentioned (3).

EXAMPLES

Hereinafter, the constitution and effects of the present invention willbe explained in detail by showing Examples. However, this invention isnot limited only to these examples, explanation and description.

Raw materials, experimental procedures, and the like used in theseexamples are as follows.

(1) Pulmonary Surfactant

The pulmonary surfactant used as the “antigen-drug vehicle (the ADvehicle) is a sample prepared from bovine lung by the method of Howgood,et al. (PSF-1) (Howgood S, et al.,: Effects of a surfactant-associatedprotein and calcium ions on the structure and surface activity of lungsurfactant lipids. Biochemistry, 24, 184-190, 1985), or largely a sampleprepared by extracting the former sample with 1-butanol to removewater-soluble protein ingredients, SP-A and SP-D, or reduce them todetection limit or less (PSF-2) (Haagasman H P, et al.,: The major lungsurfactant protein, SP28-36, is a calcium-dependent, carbohydratebinding protein, J. Biol. Chem. 262, 13977-13880, 1987), and further anysynthetic peptide of the active regions of the lipid-soluble proteins,SP-B or SP-C, which contains 40 wt. % or more to the whole ofphospholipid comprising mainly phosphatidyl choline and dipalmitoylphosphatidyl choline, and further contains 10 to 20% ofphosphatidylglycerol and 2 to 5% of phosphatidyl serine, which aresimilar to the pulmonary surfactant lipid, or a sample containing 0 to3.5% of both peptides in combination. Among these samples, investigatedwere a sample containing both peptides in combination of the activeregions of SP-B and SP-C (PSF-3), a sample containing a syntheticpeptide of the active region of SP-B (PSF-4) and a sample containing asynthetic peptide of the active region of SP-C (PSF-5). Alternatively,known samples corresponding to PSF-1 or PSF-2, for example, Survanta,Infasurf, Curosurf, Humansurf, Exosurf, Alveofact, and the like(trademark) are also used.

(2) Animal

6 week-old female BALB/c mouse, and 10 week-old Hartley guinea pig werepurchased from Japan SLC, Inc. (Shizuoka, Japan) and used. All of theanimal experiments were conducted in the cage for infected animals (P2level) of the experimental animal center of the medical faculty ofUniversity of Tokushima, and conducted in accordance of the guideline ofthe animal experiment committee of the medical faculty of University ofTokushima.

(3) Preparation of Split-Type Influenza Vaccine

Preparation of split-type influenza vaccine was conducted with thefollowing procedures using the suspension derived from a grown egg wherethe influenza virus, A Aichi/68/2/H3N2 strain was inoculated (1×10⁸Plaque forming unit [PFU]) (supplied by Professor Masanobu Ouchi inmicrobiology class of Kawasaki medical school). P-propiolactone (WakoPure Chemical Industries, Ltd., Osaka, Japan) was added to the virussuspension, which was dialyzed in 0.004 M PBS (TakaraBio, Inc., Shiga,Tokyo, Japan) overnight, to 0.05% of the liquid amount and 8 nM of thefinal concentration, and incubated for 18 hours in an ice bath. Then, itwas incubated for 1.5 hours at 37° C. to hydrolyze the β-propiolactone.Then, Tween 20 (Wako Pure Chemical Industries, Ltd.) was added to 0.1%of the final concentration, and diethylether (Wako Pure ChemicalIndustries, Ltd.) was further added in the equal amount to that ofTween, and mixed with shaking for two hours at 4° C. This solution wascentrifuged at 2,000 rpm for 5 minutes to collect the aqueous layer.Furthermore, removal of diethylether was conducted from the aqueouslayer with Automatic Environmental SpeedVac System (SAVANT INSTRUMENTS,INC., New York, USA). This was filtered with a 0.45 μm filter(MILLIPORE, Massachusetts, USA), to prepare inactivated split-typeinfluenza vaccine.

(4) Immunization

To the split-type influenza vaccine prepared by the production methoddescribed above were mixed and used the above-mentioned pulmonarysurfactant (PSF-1), 1-butanol extracted pulmonary surfactant (PSF-2),the synthetic peptides of the active regions of SP-B and SP-C and thepulmonary surfactant lipid (PSF-3, -4 and -5), known pulmonarysurfactant product, or cholera toxin B subunit (CTB, SIGMA, Missouri,USA) as an immune adjuvant. The pulmonary surfactant or thecorresponding sample described above was suspended in PBS in situ at aconcentration required in vaccine administration, and treated withultrasonic wave for 5 minutes at room temperature to give a uniformsuspension. To this was added 0.1 μg of the split-type influenza vaccineto 0.1 μg by dry weight of the pulmonary surfactant or the correspondingsample, and mixed in a vortex mixer, and left for 1 hour at roomtemperature to be used. CTB was adjusted in situ in the similar manner,and mixed in 0.1 μg to 0.1 μg of the split-type influenza vaccine(Watanabe I, et al.,: Characterization of protective immune responsesinduced by nasal influenza vaccine containing mutant cholera toxin as asafe Adjuvant (CT112K). Vaccine 2002; 20: 3443-55).

For nasal administration of the vaccine, the above-mentioned adjustedproduct was diluted in PBS to be a 0.1 μg/1 μl solution of Phosphatebuffered saline (PBS), and this was administered to both sides by 1 μl,respectively per one animal and 2 μl in total was nasal-administered toboth sides of the nasal cavities of the mouse anesthetized with ether.For conventional subcutaneous injection, which was conducted forcomparison, the split-type influenza vaccine was diluted in PBS to be asolution of 0.1 μg/50 μl, and this was administered to the hypoderma ofthe mouse neck. For the control group, PBS was administered in the sameamount as that of the vaccine solution. After 4 weeks, secondimmunization was carried out in the same manner as the firstimmunization, and two weeks after the second immunization, the washingsolutions of the nasal cavity and the alveoli, and the serum of themouse, were prepared, which were used in measurement of virus-specificIgA and IgG, and virus infection experiment.

(5) Preparation of the Washing Solutions of the Nasal Cavity and theAlveoli, and the Serum of the Mouse

The vaccine-administered mouse was cut open in the abdomen and the chestunder pentobarbital anesthesia, and the trachea was cut open, and Atomvenous catheter tapered 3 Fr (Atom medical Corporation, Tokyo, Japan)was inserted into the lung, and 1 ml of physiological saline solutionwas infused, and this solution was collected. This was repeated threetimes, and the collected solution, 3 ml in total was used as the washingsolution of the alveoli. After collecting the washing solution of thealveoli, the atom vein catheter was inserted from the opened trachea tothe direction of the nasal cavity, and 1 ml of physiological salinesolution was infused, and the solution coming from the nose wascollected. This solution was used as the washing solution of the nose.Furthermore, blood collecting was conducted from the heart, and theserum was prepared by centrifuge at 5,000 rpm for 10 minutes.

(6) Quantitization of Protein

The protein content in the washing solutions of the nose and the lung,and the serum was measured with BCA Protein Assay Reagent Kit (PIERCE,Illinois, USA) (Smith P K., et al.,: Measurement of protein usingbicinchoninic acid. Anal. Biochem., 150, 76-85, 1985). Absorbance at 562nm was measured with SPECTRAmax PLUS 384 (Molecular Devices Corporation,California, USA).

(7) Experiment for Virus Infection and Evaluation for Infection Value.

The influenza virus strain, A Aichi/68/2/H3N2 strain, which is the sameas the virus strain used in the preparation of the split-type influenzavaccine, was used in infection. Two weeks after completion of the secondimmunization, the mouse was anesthetized with ether, and a suspensionderived from an egg where the influenza virus was grown, was dropped forinfection into both sides of the nasal cavities in 7×10⁴ PFU/3 μl. 3weeks after the infection, the washing solutions of the nasal cavity andthe alveoli were prepared in the same manner as described above, whichwere used in evaluation of virus infection value. The evaluation ofvirus infection value was conducted using A 549 cells (supplied byProfessor Masanobu Ouchi in microbiology class of Kawasaki medicalschool). A 549 cells were incubated under the condition of 5% bovinefetal serum/DMEM (Gibco, New York, USA). A 549 cells were subcultured tobe 100% confluent in 6 well-incubation plate (Greiner DeutscheStuttgart), and changed to no-serum medium after 24 hours. The washingsolutions of the nasal cavity and the alveoli of influenza infectionmouse were dropped in 500 μl, respectively to each well, and incubatedin CO₂ incubator at 37° C. for 12 hours to 16 hours. To this was addedthe 1% PBS solution of erythrocyte, which was collected from the guineapig, and the mixture was left for 5 minutes at room temperature. Themixture was washed with 1 mM Ca²⁺/Mg²⁺ PBS, and evaluation of virusinfection value was conducted by counting the cell agglutinatingerythrocyte as a virus infection cell (Tashiro M., Homma M.:Pneumotropism of Sendai virus in relation to protease-mediatedactivation in mouse lungs. Infect. Immun. 39, 879-888, 1983).

(8) Purification of Anti-Influenza Specific IgA and IgG

Purification of anti-influenza specific IgA and IgG was conducted asfollows to be used as standard for quantitization in ELISA assay. IgGfractions were purified from the lung-washing solutions of the influenzavaccine-administered and virus-infected mice by affinity chromatographywith recombinant E. coli expression Protein G cephalose 4B column (ZYMEDLABORTORIES INC, San Francisco, USA). Anti-mouse IgA goat IgG (SIGMA)was bound to BrCN-activated cephalose 4B column (Amersham Bioscience,New Jersey, USA), and using this, IgA fractions were purified byaffinity chromatography from the Protein G passing-through fraction. Topurify virus-specific antibody from these IgG and IgA fractions,inactivated split-type influenza vaccine used in immunization was boundto BrCN activated cephalose column, and anti-influenza specific IgA andIgG were purified, respectively by antigen affinity chromatography fromthe IgG and IgA fractions using this. Coupling of the split-typeinfluenza protein as a ligand to the column was carried out by thebinding reaction with 0.1 M NaHCO₃/0.5 M NaCl buffer solution (pH 8.5),and removing the free ligand with 0.1 M acetic acid/0.5 M NaCl buffersolution (pH 8.5) and neutralizing by PBS (pH 7.5). Afteraffinity-binding reaction by PBS (pH 7.5) and removal of free antibody,each of the affinity chromatography was subjected to elution of thespecific antibody by glycine-HCl buffer solution (pH 2.8). Elutedfractions were neutralized immediately by 0.5 M Tris HCl buffer solution(pH 9.0), and dialyzed with MilliQ water and lyophilized, which was usedas dissolved in PBS in situ.

(9) Quantitization of Anti-Influenza Antibody

Contents of anti-influenza IgA and IgG in the washing solutions of thenasal cavity and the alveoli, and the serum, were quantitized by ELISAassay. The ELISA assay was carried out according to the method of MouseELISA Quantitization kit of BETHYL LABORATORIES (Texas, USA). To eachwell of 96 well Nunc immunoplate (Nalgen Nunc International, New York,USA), 1 μg of the vaccine and 100 μl of 1 μg/ml PBS solution of bovineserum albumin (BSA, SIGMA, Missouri, USA) were added, and reaction forlayer-solidification was conducted at 4° C. overnight. Then, each wellwas washed three times with the washing solution (50 mM Tris, 0.14 MNaCl, 0.05% Tween 20, pH 8.0) to remove the vaccine solution. To eachwell was added 50 mM Tris-HCl buffer solution containing 0.15 M NaCl and1% BSA, and the blocking reaction was carried out at room temperaturefor 1 hour. Each well was washed three times with the washing solution,and then added were 100 μl of the washing solutions of the nasal cavityand the alveoli, and the serum, which had been diluted with a suitableamount of a sample-binding buffer solution (50 mM Tris, 0.15 M NaCl, 1%BSA, 0.05% Tween 20, pH 8.0), and the mixture was left for reaction atroom temperature for 2 hours. Color reaction was conducted with TMBMicrowell Peroxidase Substrate System (Kirkegaard & Perry Laboratories,Inc. Maryland, USA), using Goat anti-mouse IgA or IgG-horse rADishperoxidase (HRP) (BETHYL LABORATORIES INC.) as secondary antibody. Thereaction was terminated by adding 100 μl of 2 M H₂SO₄ (Wako PureChemical Industries, Ltd.) to each well, and absorbance at 450 nm wasmeasured with SPECTRAmax PLUS 384. Anti-influenza 10 ng of IgA and IgGpurified from the above-mentioned lung-washing solution was treated inthe same manner as a standard for quantitization, and obtainedabsorbance was used.

(10) Preparation of Dendritic Cells and Flow Cytometry

Preparation of dendritic cells was carried out from the nose, lung andspleen collected from the mice of each group (4 in one group) 2 daysafter the first immunization, by the method of Gonzalez-Juarrero M(Gonzalez-Juarrero M, Orme I M.: Characterization of murine lungdendritic cells infected with Mycobacterium tuberculosis. Infect Immun.2001; 69: 1127-33). Preparation of dendritic cells from the nose andcollagenase treatment therefor was conduced according to the method ofAsanuma H, et al. (Asanuma H, et al.,: Characterization of mouse nasallymphocytes isolated by enzymatic extraction with collagenase. J.Immunol. Methods 1995; 187: 41-51). The dendritic cells prepared fromeach of the tissues, were washed with 1 mM EDTA/PBS, and added per 10⁶cells were each 1 μg/ml of FITC conjugated Anti-IA/IE (MHC class II) andPE conjugated Anti CD40 or FITC conjugated Anti-CD80 (B7-1) and PEconjugated Anti CD86 (B7-2) (BD Bioscience, New Jersey, USA), and leftfor reaction for 30 minutes on the ice as 50 μl 1 mM EDTA/PBSsuspension. Free antibody was removed by conducting washing twice with 1ml of 1 mM EDTA/PBS, and the cells were resuspended in 1 ml of 1 mMEDTA/PBS. Using this, detection for the modification factor on the cellsurface was conducted by BD FACS Callibur (BD Bioscience).

(11) Quantitization of TGF-β1

Secretion amount of TGF-β1 in the washing solutions of the nasal cavityand the alveoli was quantitized by ELISA assay. The ELISA assay wasconducted using TGF-β1 ELISA kit (BIOSOURCE INTERNATIONAL, California,USA) according to the instruction attached to the kit.

(12) Quantitization of Various Cytokines

The amounts of cytokines secreted, respectively in the washing solutionsof the nasal cavity and the alveoli and from the lymphocytes of thespleen (Interleukin 4: IL-4, IL-5, IL-6 and IL-13), were quantitized bymarketed ELISA kit.

(13) Preparation of PSF-3 Comprising Synthetic Peptides of SP-B and SP-Cand Phospholipid

Each peptide of SP-B 253-278 (SEQ ID No. 16) and SP-C 24-58 (SEQ ID No.21) was chemically synthesized by known method. These peptides wereadded to a phospholipid membrane (dipalmitoyl phosphatidyl choline (75),phosphatidylglycerol (25) and palmitic acid (10)) to prepare aphospholipid membrane of plate form, and AD vehicle PSF-3 was prepared.

Example 1 Comparison of Nasal Influenza Vaccine andSubcutaneous-Injection Influenza Vaccine on Virus Growth InhibitionAction

As the nasal vaccine, 0.1 μg of the split-type influenza vaccine wasadministered to both noses of BALB/c mouse by 1 μl, respectively as aPBS solution, alone or with 0.1 μg of 1-butanol-extracted (excludingSP-A and SP-D) surfactant (PSF-2 below) as the “AD vehicle”, or 0.1 μgof CTB. As the subcutaneous-injection vaccine, the vaccine in the sameamount as that of the nasal vaccine was administered as 50 μl PBSsolution in total to the hypoderma of the neck of BALB/c mouse, alone orwith adding PSF-2 as the AD vehicle or CTB as an adjuvant. After 4weeks, second immunization was conducted in the same manner as in thefirst immunization. To the control group, same amount of PBS wasadministered, respectively. 2 weeks after the second immunization,influenza virus of 6.6×10⁴ PFU was subjected to nasal infection as 3 μlPBS solution. 3 days after the infection, the mouse was slaughtered, andthe washing solutions of the nasal cavity and the alveoli were prepared,and using this, evaluation of virus infection value was conducted(n=15-20; average±SE; *, the significance level by T-test was p<0.01 tothe vaccine administration group)

As shown in FIGS. 1 (a) and (b), in case of nasal influenza vaccineadministration, growth of the influenza virus in the washing solutionsof the nasal cavity and the alveoli was inhibited even by theindependent vaccine administration, but PSF-2 or CTB reinforced theeffects significantly and growth of the influenza was nearly perfectlyinhibited, ensuring the effects of the vaccine. Though now shown in thefigure, in the case of using PSF-1 or PSF-3 instead of PSF-2, similareffects were obtained. Similar phenomena were also found for PSF-4 and-5, but the effects were attenuated.

Though now shown in the figure, even in case that PSF-2 or CTB werenasally administered as independent, respectively in the firstimmunization and the second immunization, inhibitory effects for virusgrowth were not found in either case, and thus the effects of PSF-2 orCTB were determined as reinforcement effects for the vaccine effects.

On the other hand, in case of subcutaneous-injection influenza vaccine,as shown in (c) and (d) of FIG. 1, the titer (PFU) of the influenzavirus in the washing solutions of the nasal cavity and the alveoli wasreduced significantly even with the independent vaccine, so the effectsof the vaccine was found, but reinforcement effects of PSF-2 or CTB werenot found. In other words, in case of subcutaneous administration,nearly no or very little reinforcement effects of PSF-2 or CTB onimmunity were found. Though now shown in the figure in this experiment,even in case that PSF-2 or CTB is subcutaneously administered asindependent, respectively in the first immunization and the secondimmunization, inhibitory effects for virus growth were not found ineither case. Though now shown in the figure, in the case of using PSF-1,PSF-3, PSF-4 and PSF-5 instead of PSF-2, reinforcement effects on thevaccine action were not found.

Example 2 Influence of PSF-2 or CTB on Production of Anti-InfluenzaSpecific Antibodies (IgA and IgG) in the Washing Solution of the NasalCavity After (a) nasal and (b) Subcutaneous-Injection Influenza VaccineAdministration

In the same manner as described in FIG. 1, as the nasal vaccine, 0.1 μgof the split-type influenza vaccine was administered to both noses ofBALB/c mouse by 1 μl, respectively, i.e., 2 μl in total as a PBSsolution, alone or with 0.1 μg of PSF-2 as the “AD vehicle”, or 0.1 μgof CTB as an adjuvant. As the subcutaneous vaccine, the vaccine in thesame amount as that of the nasal vaccine, PSF-2 or CTB was administeredas 50 μl PBS solution to the hypoderma of the neck of BALB/c mouse.After 4 weeks, second immunization was conducted in the same manner asin the first immunization. To the control group, same amount of PBS wasadministered, respectively. 2 weeks after the second immunization,influenza virus of 6.6×10⁴ PFU was subjected to nasal infection as 3 μlPBS solution. 3 days after the infection, the mouse was slaughtered, andthe washing solution of the nasal cavity was prepared, and using this,evaluation of virus infection value was conducted (n=15-20; average±SE;*, the significance level by T-test was p<0.01 to the vaccineadministration group).

The results are shown in FIGS. 2 (a) and (b). By the split-typeinfluenza vaccine nasally administered, anti-influenza specific IgA wasselectively produced in the nasal cavity, and thus increased in thewashing solution, but PSF-2 or CTB increased the amount of this specificIgA in equivalent extent remarkably. On the other hand, even in case ofthe subcutaneous injection, increase of specific IgA amount in thewashing solution of the nasal cavity by independent split-type influenzavaccine was found, but the extent was low compared to that of the nasaladministration. In addition, in case of the subcutaneous injection ofthe vaccine, immunity reinforcement effects of PSF-2 or CTB were notfound in either case in production of IgA or IgG. Though now shown inthe figure, in the case of using PSF-1 or PSF-3 instead of PSF-2,similar effects were obtained. Similar phenomena were also found forPSF-4 and -5, but the effects were attenuated.

Example 3 Influence of PSF-2 or CTB on Production of Anti-InfluenzaSpecific Antibodies (IgA and IgG) in the Washing Solution of the Lung byInfluenza Vaccine of (a) Nasal Administration and (b)Subcutaneous-Injection

As the nasal vaccine, 0.1 μg of the split-type influenza vaccine wasadministered to both noses of BALB/c mouse by 1 μl, respectively, i.e.,2 μl in total as a PBS solution, alone or with 0.1 μg of PSF-2 as the“AD vehicle”, or 0.1 μg of CTB as an adjuvant. As the subcutaneousvaccine, the vaccine in the same amount as that of the nasal vaccine,PSF-2 or CTB was administered as 50 μl PBS solution to the hypoderma ofthe neck of BALB/c mouse. After 4 weeks, second immunization wasconducted in the same manner as in the first immunization. To thecontrol group, same amount of PBS was administered, respectively. 2weeks after the second immunization, influenza virus of 6.6×10⁴ PFU wassubjected to nasal infection as 3 μl PBS solution. 3 days after theinfection, the mouse was slaughtered, and the washing solution of thelung was prepared, and using this, influence of the antigen-drug vehicleon production of anti-influenza specific antibodies (IgA and IgG) wasinvestigated (n=15-20; average±SE; *, the significance level by T-testwas p<0.01 to the vaccine administration group).

As shown in FIGS. 3 (a) and (b), promoting effects of PSF-2 or CTB onproduction of anti-influenza specific antibodies were remarkable,similarly to the case of nasal administration of the vaccine, and therewas no big difference between them. This immunity reinforcement effectswere specific for IgA, and not found for IgG. In case of thesubcutaneous injection, increase of IgA in the washing solution of thelung, but immunity reinforcement effects of PSF-2 or CTB were not foundsimilarly to the case of the washing solution of the nasal cavity.Though now shown in the figure in this experiment, even in case thatPSF-2 or CTB was administered nasally or subcutaneously as independent,respectively in the first immunization and the second immunization,increase of virus-specific IgA or IgG in the washing solution of thelung was not found in either case, and thus the effects of PSF-2 or CTBwere determined as reinforcement effects for the vaccine actions. Thoughnow shown in the figure, in the case of using PSF-1 or PSF-3 instead ofPSF-2, similar effects were obtained. Similar phenomena were also foundfor PSF-4 and -5, but the effects were attenuated.

Example 4 Influence of PSF-2 or CTB on Production of Anti-InfluenzaSpecific Antibodies (IgA and IgG) in the Blood by Influenza Vaccine of(a) Nasal Administration and (b) Subcutaneous-Injection

As the nasal vaccine, 0.1 μg of the split-type influenza vaccine wasadministered to both noses of BALB/c mouse by 1 μl, respectively, i.e.,2 μl in total as a PBS solution, alone or with 0.1 μg of PSF-2 as the“AD vehicle”, or 0.1 μg of CTB as an adjuvant. As the subcutaneousvaccine, the vaccine in the same amount as that of the nasal vaccine,PSF-2 or CTB was administered as 50 μl PBS solution to the hypoderma ofthe neck of BALB/c mouse. After 4 weeks, second immunization wasconducted in the same manner as in the first immunization. To thecontrol group, same amount of PBS was administered, respectively. 2weeks after the second immunization, the mouse was slaughtered, bloodcollection from the heart was conducted, and the serum was prepared fromthis, and quantitization of the expression amount of anti-influenzaantibodies was conducted using this (n=15-20; average±SE).

As shown in FIGS. 4 (a) and (b), for the production amount of bloodanti-influenza antibodies IgA (white bar) and IgG (black bar), slightincrease of IgA or IgG was found in nasal vaccine administration, butincrease of IgA or IgG by PSF-2 or CTB was not found, and thus iummunityreinforcement effects were not found. On the other hand, in case of thesubcutaneous injection, remarkable increase of IgG and definite increaseof IgA were found by the split-type influenza vaccine, but also in thiscase, increase of antibody production by PSF-2 or CTB was not found andthus immunity reinforcement effects were not found. Particularly, incase of the subcutaneous injection, IgG increased specifically in blood.In the case of using PSF-1 instead of PSF-2, nearly similar effects wereobtained (not shown in the figure).

In addition, even in case that PSF-2 or CTB was administered nasally orsubcutaneously as independent, respectively in the first immunizationand the second immunization, increase of virus-specific IgA or IgG inblood was not found, and thus the effects of PSF-2 or CTB weredetermined as reinforcement effects for the vaccine actions (not shownin the figure).

Example 5 Influence of PSF-2 or CTB on Antigen-Presenting Ability ofDendritic Cells of the Nose, Lung or Spleen by Influenza Vaccine of (a)Nasal Administration and (b) Subcutaneous-Injection

As the nasal vaccine, 0.1 μg of the split-type influenza vaccine wasadministered to both noses of BALB/c mouse in 2 μl in total as 1 μl PBSsolution, alone or with 0.1 μg of PSF-2, or 0.1 μg of CTB. As thesubcutaneous vaccine, the split-type influenza vaccine in the sameamount as that of the nasal vaccine, PSF-2 or CTB was administered as 50μl PBS solution to the hypoderma of the neck of BALB/c mouse. After 2days, the mouse was slaughtered, and the dendritic cells were preparedfrom the nose, lung or spleen, and expression level of MHC class II,CD40, B7-1 (CD80) and B7-2 (CD80) on the cell surface was measured byflow cytometry.

As a result, increase of expression of antigen-presenting relatedmolecules, CD40 and B7-2 was found on the membrane surface of thedendritic cells (antigen-presenting cells) in the nose, where thevaccine had been challenged by CTB, and adjuvants effects were found inmolecular level. In case of PSF-2, increase of expression of MHC IImolecule was also found in addition to CD40 and B7-2 of the dendriticcells in the nose, and it was shown that at least three molecules of thedendritic cells are involved in the immunity reinforcement effects.

However, definite change was not found in CD40, B7-2 or MHC II moleculeof the dendritic cells in the lung and spleen. In the case of usingPSF-1 or PSF-3 instead of PSF-2, nearly similar effects were obtained.Similar phenomena were also found for PSF-4 and -5, but the effects wereattenuated.

Example 6 Influence of SPF-2 or CTB on TGF-β1 Secretion Level in theMucosal Membrane of (a) the Nasal Cavity and (b) the Alveoli by NasalAdministration of Influenza Vaccine

As the nasal vaccine, 0.1 μg of the split-type influenza vaccine wasadministered to both noses of BALB/c mouse in 2 μl in total as 1 μl PBSsolution, alone or with 0.1 μg of SPF-2, or 0.1 μg of CTB. As thesubcutaneous vaccine, the vaccine in the same amount as that of thenasal vaccine, PSF-2 or CTB was administered as 50 μl PBS solution tothe hypoderma of the neck of BALB/c mouse. After 4 weeks, secondimmunization was conducted in the same manner as in the firstimmunization. To the control group, same amount of PBS was administered,respectively. 2 weeks after the second immunization, the mouse wasslaughtered, and the washing solution of the nasal cavity was prepared,and using this, quantitization of TGF-β1 secretion amount was conducted(n=15-20; average±SE; *, the significance level by T-test was p<0.01 tothe vaccine administration group).

It has been known that for differentiation of B cell to IgA-productioncell (class switch), local TGF-β1 concentration present in theproduction cell is important (Stavnezer, J.: Regulation of antibodyproduction and class switching by TGF-beta. J. Immunol. 155(4),1647-1651, 1995). Herein, TGF-β1 concentration in a local mucosalmembrane of (a) the nasal cavity or (b) the alveoli was investigated.

The TGF-β1 concentration in the local mucosal membrane of the nasalcavity or the alveoli where the split-type influenza vaccine wasadministered, increased significantly in presence of SPF-2 or CTB. Theextent of increase was such that there was no significant differencebetween SPF-2 and CTB. The results are shown in FIGS. 5( a) and (b). Itwas found that SPF-2, which was derived from the living body, increasedTGF-β1 concentration which urges promotion of differentiation of B cellsecreting IgA as much as CTB, which is exogenous toxin. Though not shownin the figure, in the case of using PSF-1 or PSF-3 instead of PSF-2,nearly similar effects were obtained. Similar phenomena were also foundfor PSF-4 and -5, but the effects were attenuated. In addition, even incase that PSF-2 or CTB was administered nasally or subcutaneously asindependent, respectively in the first immunization and the secondimmunization, increase of TGF-β1 concentration was not found, and thusthe effects of PSF-2 or CTB were determined as reinforcement effects forthe vaccine actions (not shown in the figure).

Example 7 Influence of PSF-2 or CTB on Production of Anti-InfluenzaSpecific Antibodies (IgA and IgG) in (a) the Nasal Cavity, (b) theAlveoli and (c) the Blood by Nasal Administration of Influenza Vaccine

As the nasal vaccine, 0.2 μg of the split-type influenza vaccine wasadministered to both noses of BALB/c mouse by 1 μl, respectively, i.e.,2 μl in total as a PBS solution, alone or with 0.2 μg of PSF-2 as the“AD vehicle”, or 0.2 μg of CTB as an adjuvant. After 4 weeks, secondimmunization was conducted in the same manner as in the firstimmunization. To the control group, same amount of PBS was administered,respectively. 2 weeks after the second immunization, the mouse wasslaughtered in each group, and the washing solutions of the nasal cavityand the alveoli, and serum by blood collection from the heart wereprepared, and using these, quantitization of expression amount ofanti-influenza antibody was conducted (n=15-30; average±SE; +, p<0.01vs. the vaccine independent administration).

As shown in FIGS. 6 (a) and (b), in the washing solutions of the nasalcavity and the alveoli, blood anti-influenza antibody IgA (blue circlet)showed remarkable increase in case that PSF-2 and the vaccine wereadministered. However, as shown in FIG. 6 (c), increase of IgG (redcirclet) in blood was not found.

On the other hand, in case that CTB and the vaccine were administered,IgG and IgG increased in the washing solutions of the nasal cavity andthe alveoli (FIGS. 6 (a) and (b)), and also remarkably in blood (FIG. 6(c)).

As described above, in case of CTB, it reacted with the antigeninoculated nasally, and caused systemic immune response, as well asestablishment of local immunity, as reported conventionally. On theother hand, in case of PSF-2, it only established local mucosalimmunity.

In addition, J. Freek van Iwaarden, et al. (Non-Patent Document 4) havereported that if macrophage is removed artificially from the lung, SP-Band lipid can induce systemic immune reaction, but in the case thatmacrophage is not removed, they cannot induce immunity. In addition, inthis document, the amount of SP-B plus lipid necessary for induction ofthe systemic immune reaction, is 250 to 300 μl, which is quite differentfrom the PSF-2 administration amount in the above-mentioned Examples(0.2 μl). However, it has never mentioned local mucosal immunity.

Example 8 Influence of SPF-2 or CTB on Various Cytokines Secreted Fromthe Nose, Lung or Lymphocyte of Spleen by Nasal Administration ofInfluenza Vaccine

As the nasal vaccine, 0.2 μg of the split-type influenza vaccine wasadministered to the upper respiratory tract of BALB/c mouse in 2 μl intotal as 1 μl PBS solution, alone or with 0.2 μg of PSF-2 or 0.2 μg ofCTB. 2 weeks after the second immunization, the mouse was slaughtered,and quantitization of secretion amount of TGF-β1 and cytokines (IL-4,IL-5, IL-6 and IL-13) from the nose, lung or lymphocyte of spleen wasconducted (n=15-20; average±SE; +++, p=0.06; ++, P=0.05; +, P=0.01 vs.the vaccine independent administration).

As shown in FIGS. 7 (a) to (e), TGF-β1, IL-5 and IL-6 were found toincrease significantly in secretion, whereas IL-4 and IL-13 were foundto increase significantly in the local mucosal membrane (nose, lung)after the vaccine immunity along with PSF-2. On the other hand,significant increase of any cytokine was not observed in the spleen.

From the above results, it was found that PSF-2 increase Th2-typecytokine, which promotes differentiation and induction of B cellproducing IgA.

Example 9 Influence of PSF-3 on Production of Anti-Influenza SpecificAntibodies (IgA and IgG) in (a) the Nasal Cavity, (b) the Alveoli and(c) the Blood by Nasal Administration of Influenza Vaccine

As the nasal vaccine, 0.2 μg of the split-type influenza vaccine wasadministered to both noses of BALB/c mouse by 1 μl, respectively, i.e.,2 μl in total as a PBS solution, alone or with 0.2 μg of PSF-3 (amixture of same amount of SP-B fragment, i.e., 253 to 278 peptidesdescribed in SEQ ID No. 16, and SP-C fragment, i.e., 24 to 58 peptidesdescribed in SEQ ID No. 21) as the “AD vehicle”, or 0.2 μg of lipidingredient. After 4 weeks, second immunization was conducted in the samemanner as in the first immunization. To the control group, same amountof PBS was administered, respectively. 2 weeks after the secondimmunization, the mouse was slaughtered in each group, and the washingsolutions of the nasal cavity and the alveoli, and serum by bloodcollection from the heart were prepared, and using these, quantitizationof expression amount of anti-influenza antibody was conducted (n=15-30;average±SE; +, p<0.01 vs. the vaccine independent administration).

As shown in FIGS. 8 (a) and (b), in the washing solutions of the nasalcavity and the alveoli, blood anti-influenza antibody IgA (blue circlet)showed remarkable increase in case that PSF-3 and the vaccine wereadministered, but significant increase of IgG (red circlet) was notfound. On the other hand, in serum (c), neither IgA (blue circlet) norIgG (red circlet) was found to increase significantly.

INDUSTRIAL APPLICABILITY

The “AD vehicle” according to the present invention, exerts a functionof transporting all the substances such as antigen, drug, nutrient, andthe like from the mucosal membrane of the nose, trachea, intestine, andthe like, or the skin into cells, and also induces preferential andselective production of IgA, enabling a mucosal vaccine, prevention andtreatment of allergy, transmucosal and transdermal DDS, and transmucosaland transdermal administration of useful substance such as a drug,nutrient, and the like. Furthermore, it has been approved already forits clinical use and safety in this country or other countries.

Accordingly, application and use of the “AD vehicle” is expected in verybroad range of industries such as medication/pharmaceutics in biologicalpreparations, DDS, and the like, food and drink industries in functionalfood, health food, and the like, agriculture and agricultural chemicalsin raising and cultivation of agricultural products, anti-diseasemeasures, insect destruction, and the like, cultivation fishery infish-disease vaccine and administration thereof, and the like,architecture or environment preservation in anti-ant, anti-insect, andthe like.

1. A composition for inducing mucosal immunity against an influenzavirus by promoting the production of an IgA antibody against the virusin a local mucosal membrane, comprising an influenza virus split antigenand an antigen-drug vehicle which is a complex consisting of: apulmonary surfactant protein B or a peptide consisting of the amino acidsequence of SEQ ID NO: 2, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19or 20, a pulmonary surfactant protein C or a peptide consisting of theamino acid sequence of SEQ ID NO: 4, 9, 21 or 6, and at least one lipidselected from the group consisting of phosphatidyl choline, dipalmitoylphosphatidyl choline, phosphatidyl serine, dipalmitoylglycerophosphocholine, diacyl glycerophosphoglycerol,phosphatidylglycerol, phosphatidyl inositol, phosphatidyl ethanolamine,phosphatidic acid, lauric acid, myristic acid, palmitic acid, stearicacid and oleic acid, wherein the composition is suitable for nasaladministration.
 2. The composition according to claim 1, wherein theinduction of mucosal immunity is further made by promoting theproduction of TGF-β1 and Th2 type cytokine against the influenza virusin the local mucosal membrane.
 3. A method of inducing mucosal immunity,which comprises administering the composition according to claim 1, intoa nasal cavity or upper respiratory tract of a subject in need thereof.4. The method of inducing mucosal immunity according to claim 3, whereinthe induction of mucosal immunity is made by promoting the production ofan IgA antibody in the local mucosal membrane.
 5. The method of inducingmucosal immunity according to claim 3, wherein the induction of mucosalimmunity is made by promoting the production of TGF-β1 and Th2 typecytokine in the local mucosal membrane.